Deleting IP6K1 stabilizes neuronal sodium–potassium pumps and suppresses excitability

Inositol pyrophosphates are key signaling molecules that regulate diverse neurobiological processes. We previously reported that the inositol pyrophosphate 5-InsP7, generated by inositol hexakisphosphate kinase 1 (IP6K1), governs the degradation of Na+/K+-ATPase (NKA) via an autoinhibitory domain of PI3K p85α. NKA is required for maintaining electrochemical gradients for proper neuronal firing. Here we characterized the electrophysiology of IP6K1 knockout (KO) neurons to further expand upon the functions of IP6K1-regulated control of NKA stability. We found that IP6K1 KO neurons have a lower frequency of action potentials and a specific deepening of the afterhyperpolarization phase. Our results demonstrate that deleting IP6K1 suppresses neuronal excitability, which is consistent with hyperpolarization due to an enrichment of NKA. Given that impaired NKA function contributes to the pathophysiology of various neurological diseases, including hyperexcitability in epilepsy, our findings may have therapeutic implications.

While pharmacological inhibition of NKA by cardiac glycosides such as digoxin is well-established, there are limited clinical options for therapeutically activating NKA.Boosting NKA levels and/or activity has demonstrated benefits for treating certain neurological disorders such as epilepsy and Parkinson disease.NKA mutations and pharmacological inhibition promote cellular hyperexcitability and drive epilepsy, whereas NKA-activating antibodies were shown to attenuate seizure susceptibility [17].NKA deficiency aggravates α-synuclein pathology, whereas therapeutic NKA-stabilizing antibodies were shown to ameliorate α-synuclein pathology [18].Although insulin and β2 adrenergic receptor agonists are used clinically to treat acute hyperkalemia by indirectly increasing NKA expression, they exhibit significant side effects due to their nonspecific mechanisms [19][20][21].Recent preclinical studies have demonstrated therapeutic effects of small-molecule IP6K inhibitors [22][23][24], which may be a targeted strategy to boost neuronal NKA levels for treating neurological disorders such as epilepsy or Parkinson disease.
In this work, we expand the physiological relevance of IP6K1 governing NKA stability by studying the brains of wild-type (WT) and IP6K1 KO mice.We isolated cerebral cortices and performed western blots (Fig. 1A).NKA levels are approximately 60% higher in cerebral cortices from IP6K1 KO mice compared to those from WT mice (Fig. 1B, P = 0.0035).We further corroborated the increase in cerebral cortex NKA levels via immunostaining (Fig. 1C, D).Our previous work on NKA in kidneys of IP6K1 KO mice revealed that the increased protein level was accompanied by an increase in total NKA activity [12].We reasoned that increased NKA levels and pumping activity in the brain would hyperpolarize neurons and suppress neuronal excitability.To characterize functional differences between WT and IP6K1 KO neurons in the cerebral cortex, we performed electrophysiological studies.The frequency of action potentials in IP6K1 KO neurons was markedly lower than that of WT neurons (Fig. 1E, F, P = 0.0039).Moreover, the hyperpolarization phase of action potentials in IP6K1 KO neurons was greater in magnitude than that of WT neurons (Fig. 1G).
Our electrophysiological data indicate that deleting IP6K1 reduces neuronal excitability, which is consistent with an enrichment of NKA levels that hyperpolarizes neurons.However, instead of a simple hyperpolarization of the resting membrane potential, we found a specific perturbation of the afterhyperpolarization (AHP) phase of the action potential.AHPs are classified as fast (< 10 ms), medium (~ 50-100 ms), or slow (~ 3-20 s)fast AHPs follow individual action potentials whereas slow AHPs follow a train of action potentials [25,26].AHPs can regulate neuronal firing rates [27].While AHPs are primarily generated by calcium-gated potassium channels, slow AHPs can be additionally mediated by NKA [26,28,29].Our results show an extended fast AHP and suggest a novel role for NKA beyond regulating slow AHPs, but further studies involving genetic or pharmacological manipulation of NKA in IP6K1 KO neurons are needed to demonstrate that the perturbations in action potentials are truly mediated by NKA.Because most studies inhibit or delete neuronal NKA, IP6K1 KO neurons are a unique system to study the physiological effect and therapeutic potential of upregulating NKA in the brain.Lastly, it would be interesting for future studies to investigate whether IP6K1 also regulates cardiac action potentials.

Animals
The IP6K1 WT and KO animals were littermates from heterozygous breeding.All procedures related to animals were performed in accordance with the ethical guidelines of Shanghai Jiao Tong University School of Medicine.Animal experiments were approved by Institutional Animal Care and Use Committee of Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine.

Immunofluorescence staining
Animals were perfused and fixed with 4% paraformaldehyde.The brain slices were cut at 25 μm thickness.Slides were washed three times with TBST and blocked with 3% BSA containing 1% goat serum and 0.3% Triton X-100 for 60 min at R.T.. Then the slides were incubated with primary antibodies at 4 °C overnight and washed multiple times with TBST at R.T. Fluorescent-dye conjugated secondary antibodies were added on the slides and incubated for 1 h at R.T.. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific) for 5 min.Slices were mounted with ProLong Gold Antifade Mountant (Thermo Fisher Scientific).Images were taken under a confocal microscope (Zeiss LSM 800).
Recording electrodes (3-5 MΩ resistance) used in this experiment were pulled using P-97 horizontal puller (Sutter, USA).Intra-pipette solution contains 150 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 0.25 mM EGTA, and 5 mM MgATP adjusted to pH 7.2 with KOH.During the entire recording process, the brain slices were completely immersed into ASCF at R.T. and irrigated at a rate of 3 ml/min.The membrane was impaled to form wholecell configuration after establishing a gigaseal (> 2GΩ).Membrane capacitance and series resistance were compensated by 60-80%.Leakage and capacity currents were also subtracted on-line using a P/4 protocol.Then, the membrane potential was recorded under current clamp mode.The resting membrane potential of IP6K1 WT and KO neurons were about -64.42 ± 0.36 mV (P > 0.05).Action potential was caused and recorded after KCl was added to the recording bath.Digital signals were obtained with MultiClamp 700B amplifier (Molecular Devices, USA) and Digidata 1440 interface (Molecular Devices, USA).Meanwhile, the recording signals were filtered at 2 kHz and digitized at 10 kHz.All electrophysiology data were acquired and analyzed using pCLAMP10.0software (Molecular Devices, USA).

Statistical analysis
GraphPad Prism 9 Software (GraphPad Software Inc., La Jolla, CA) and unpaired two-tailed Student's t-test were used to perform the statistical analysis.Data are presented as mean ± SEM.The statistical level was 0.05, *P < 0.05, ** P < 0.01.

Fig. 1
Fig. 1 IP6K1 increases the expression of Na + /K + -ATPase-α1, decreases action potential frequency, and expands the hyperpolarization phase in the cerebral cortex of mice.A, B Western blot and quantification of Na + /K + -ATPase-α1 protein expression in the cerebral cortex of WT and IP6K1 KO mice.Na + /K + -ATPase-α1 expression was increased in IP6K1 KO mice (n = 3; normalized to GAPDH).C Immunostaining showed that Na + /K + -ATPase-α1 expression of neuronal cells in IP6K1 KO mice brains was increased.Neuronal nuclei antigen (NeuN) stained mature neuronal cells, and Hoechst 33342 labeled nuclei.Scale bars, 10 μm.D Line intensity plots of marked neurons in (C) showed that Na + /K + -ATPase-α1 expression was increased in the membrane and cytoplasm of IP6K1 KO neurons.E, F IP6K1 KO neurons displayed diminished action potential frequency.Data represent mean ± SEM, Student's t-test, n = 8 mice per group.G IP6K1 KO neurons exhibit a higher magnitude of the hyperpolarization phase of the action potential